Nuclear DNA Replication Research Articles

Genome organization and stability in mammalian pre-implantation development

A largely stable genome is required for normal development, even as genetic change is an integral aspect of reproduction, genetic adaptation and evolution. Recent studies highlight a critical window of mammalian development with intrinsic DNA replication stress and genome instability in the first cell divisions after fertilization. Patterns of DNA replication and genome stability are established very early in mammals, alongside patterns of nuclear organization, and before the emergence of gene expression patterns, and prior to cell specification and germline formation. The study of DNA replication and genome stability in the mammalian embryo provides a unique cellular system due to the resetting of the epigenome to a totipotent state, and the de novo establishment of the patterns of nuclear organization, gene expression, DNA methylation, histone modifications and DNA replication. Studies on DNA replication and genome stability in the early mammalian embryo is relevant for understanding both normal and disease-causing genetic variation, and to uncover basic principles of genome regulation.

High fidelity DNA ligation prevents single base insertions in the yeast genome

Finalization of eukaryotic nuclear DNA replication relies on DNA ligase 1 (LIG1) to seal DNA nicks generated during Okazaki Fragment Maturation (OFM). Using a mutational reporter in Saccharomyces cerevisiae, we previously showed that mutation of the high-fidelity magnesium binding site of LIG1Cdc9 strongly increases the rate of single-base insertions. Here we show that this rate is increased across the nuclear genome, that it is synergistically increased by concomitant loss of DNA mismatch repair (MMR), and that the additions occur in highly specific sequence contexts. These discoveries are all consistent with incorporation of an extra base into the nascent lagging DNA strand that can be corrected by MMR following mutagenic ligation by the Cdc9-EEAA variant. There is a strong preference for insertion of either dGTP or dTTP into 3–5 base pair mononucleotide sequences with stringent flanking nucleotide requirements. The results reveal unique LIG1Cdc9-dependent mutational motifs where high fidelity DNA ligation of a subset of OFs is critical for preventing mutagenesis across the genome.

Evidence that DNA polymerase δ proofreads errors made by DNA polymerase α across the Saccharomyces cerevisiae nuclear genome

We show that the rates of single base substitutions, additions, and deletions across the nuclear genome are strongly increased in a strain harboring a mutator variant of DNA polymerase α combined with a mutation that inactivates the 3´-5´ exonuclease activity of DNA polymerase δ. Moreover, tetrad dissections attempting to produce a haploid triple mutant lacking Msh6, which is essential for DNA mismatch repair (MMR) of base•base mismatches made during replication, result in tiny colonies that grow very slowly and appear to be aneuploid and/or defective in oxidative metabolism. These observations are consistent with the hypothesis that during initiation of nuclear DNA replication, single-base mismatches made by naturally exonuclease-deficient DNA polymerase α are extrinsically proofread by DNA polymerase δ, such that in the absence of this proofreading, the mutation rate is strongly elevated. Several implications of these data are discussed, including that the mutational signature of defective extrinsic proofreading in yeast could appear in human tumors.

A case report of a family with developmental arrest of human prokaryotic stage zygote.

To study the genetic variation leading to the arrest phenotype of pronuclear (PN) zygotes. We recruited a family characterized by recurrent PN arrest during in vitro fertilization (IVF) and intracytoplasmic sperm injection cycles (ICSI) and performed whole-exome sequencing for 2 individuals. The transcriptome profiles of PN-arrest zygotes were assessed by single-cell RNA sequencing analysis. The variants were then validated by PCR amplification and Sanger sequencing in the affected individuals and other family members. A family characterized by recurrent PN arrest during IVF and ICSI cycles were enrolled after giving written informed consent. Peripheral blood samples were taken for DNA extraction. Three PN-arrest zygotes from patient III-3 were used for single-cell RNA-seq as described. This phenotype was reproduced after multiple cycles of egg retrieval and after trying different fertilization methods and multiple ovulation regimens. The mutant genes of whole exon sequencing were screened and verified. The missense variant c. C1630T (p.R544W) in RGS12 was responsible for a phenotype characterized by paternal transmission. RGS12 controls Ca2+ oscillation, which is required for oocyte activation after fertilization. Single-cell transcriptome profiling of PN-arrest zygotes revealed defective established translation, RNA processing and cell cycle, which explained the failure of complete oocyte activation. Furthermore, we identified proximal genes involved in Ca2+ oscillation-cytostatic factor-anaphase-promoting complex (Ca2+ oscillation-CSF-APC) signaling, including upregulated CaMKII, ORAI1, CDC20, and CDH1 and downregulated EMI1 and BUB3. The findings indicate abnormal spontaneous Ca2+ oscillations leading to oocytes with prolonged low CSF level and high APC level, which resulted in defective nuclear envelope breakdown and DNA replication. We have identified an RGS12 variant as the potential cause of female infertility characterized by arrest at the PN stage during multiple IVF and ICSI.

CGAS: action in the nucleus.

As a canonical cytoplasmic DNA sensor, cyclic GMP-AMP synthase (cGAS) plays a key role in innate immunity. In recent years, a growing number of studies have shown that cGAS can also be located in the nucleus and plays new functions such as regulating DNA damage repair, nuclear membrane repair, chromosome fusion, DNA replication, angiogenesis and other non-canonical functions. Meanwhile, the mechanisms underlying the nucleo-cytoplasmic transport and the regulation of cGAS activation have been revealed in recent years. Based on the current understanding of the structure, subcellular localization and canonical functions of cGAS, this review focuses on summarizing the mechanisms underlying nucleo-cytoplasmic transport, activity regulation and non-canonical functions of cGAS in the nucleus. We aim to provide insights into exploring the new functions of cGAS in the nucleus and advance its clinical translation.

Loss of mitochondrial DNA is associated with reduced DNA content variability in Saccharomyces cerevisiae.

DNA content measurement by fluorescence-assisted cell sorting (FACS) provides information on cell cycle progression and DNA content variability. Saccharomyces cerevisiae mutants with DNA content variability that was reduced relative to wild-type strains had defects in mitochondrial DNA (mtDNA) maintenance and mitochondrial gene expression and were correlated with strains found to lack mtDNA ([ rho 0 ] cells) by genome sequencing and fluorescence microscopy. In contrast, mutants with increased variability had defects in cell cycle progression, which may indicate a loss of coordination between mtDNA and nuclear DNA replication. Thus, FACS measurement of DNA content variability can provide insight into cell-to-cell heterogeneity in mtDNA copy number.

Construction of a diagnostic model for ischemic stroke based on immune-related genes.

This study aimed to screen immune-related marker genes of ischemic stroke (IS). Two IS-related gene expression datasets were downloaded. The significantly differentially expressed genes (DEGs) and miRNAs (DEMs) between IS and control groups were selected. The differential immune cells were analysed. Weighted gene co-expression network analysis (WGCNA) was applied to analyse immune-related genes, followed by function analysis and interaction network construction. Then, key genes were further screened using optimization algorithm to construct a diagnostic model. Finally, miRNA regulatory network of several key genes was established. In total 321 DEGs and 140 DEMs were obtained. 11 immune cell types were significantly different between IS and control groups. WGCNA identified two key modules, involving 202 differential immune genes. The greenyellow module was enriched in biological processes and pathways associated with T cells, while the midnightblue module was mainly associated with apoptosis, and inflammatory response-related functions and pathways. Protein interaction network identified 10 hub nodes, such as CD8A, ITGAM and TLR4. LASSO regression selected 8 key feature genes, and a risk score model was established. Key model genes were enriched in 63 GO biological processes, such as microglial cell activation, and B cell apoptotic process, and 3 KEGG pathways, such as negative regulation of nuclear cell cycle DNA replication, and hematopoietic cell lineage. Finally, a total of 25 miRNA-target relationship pairs were obtained. This study identified some immune-related marker genes and constructed a diagnostic model based on 8 immune-related genes in IS.

KIF11 serves as a cell cycle mediator in childhood acute lymphoblastic leukemia.

To identify key gene in childhood acute lymphoblastic leukemia (ALL) through weighted gene co-expression network analysis (WGCNA), and their enriched biological functions and signaling pathways. Array data of the GSE73578 dataset, involving 46 childhood ALL samples, were acquired from the Gene Expression Omnibus (GEO) database. Hub modules associated with childhood ALL were screened out by WGCNA. Enriched biological functions and signaling pathways were then identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Hub genes were selected by overlapping those between down-regulated genes in GSE73578, GSE4698 and the hub module. Guilt by association (GBA) was adopted to verify the function of the identified KIF11 gene and to predict its target genes. Regulatory effects of KIF11 on the proliferation and cell cycle progression of ALL in vitro were determined by cytological experiments. WGCNA showed that the yellow module was the most relevant to childhood ALL treatment, containing 698 genes that were enriched in cell division, mitotic nuclear division, DNA replication and DNA repair, cell cycle, DNA replication and the P53 signaling pathway. The KIF11 gene was screened out and predicted as a cell cycle mediator in childhood ALL. Knockdown of KIF11 in ALL cells inhibited cell proliferation and arrested cell cycle progression in G2/M phase. The KIF11 gene is critical in the treatment process of childhood ALL, which is a promising therapeutic target for childhood ALL.

Virus–pathogen interactions improve water quality along the Middle Route of the South-to-North Water Diversion Canal

Bacterial pathogens and viruses are the leading causes of global waterborne diseases. Here, we discovered an interesting natural paradigm of water “self-purification” through virus–pathogen interactions over a 1432 km continuum along the Middle Route of the South-to-North Water Diversion Canal (MR-SNWDC) in China, the largest water transfer project in the world. Due to the extremely low total phosphorus (TP) content (ND-0.02 mg/L) in the MR-SNWDC, the whole canal has experienced long-lasting phosphorus (P) limitation since its operation in 2015. Based on 4443 metagenome-assembled genomes (MAGs) and 40,261 nonredundant viral operational taxonomic units (vOTUs) derived from our recent monitoring campaign, we found that residential viruses experiencing extreme P constraints had to adopt special adaptive strategies by harboring smaller genomes to minimize nucleotide replication, DNA repair, and posttranslational modification costs. With the decreasing P supply downstream, bacterial pathogens showed repressed environmental fitness and growth potential, and a weakened capacity to maintain P acquisition, membrane formation, and ribonucleotide biosynthesis. Consequently, the unique viral predation effects under P limitation, characterized by enhanced viral lytic infections and an increased abundance of ribonucleotide reductase (RNR) genes linked to viral nuclear DNA replication cycles, led to unexpectedly lower health risks from waterborne bacterial pathogens in the downstream water-receiving areas. These findings highlighted the great potential of water self-purification associated with virus–pathogen dynamics for water-quality improvement and sustainable water resource management.

Association of Mutations in Replicative DNA Polymerase Genes with Human Disease: Possible Application of Drosophila Models for Studies.

Replicative DNA polymerases, such as DNA polymerase α-primase, δ and ε, are multi-subunit complexes that are responsible for the bulk of nuclear DNA replication during the S phase. Over the last decade, extensive genome-wide association studies and expression profiling studies of the replicative DNA polymerase genes in human patients have revealed a link between the replicative DNA polymerase genes and various human diseases and disorders including cancer, intellectual disability, microcephalic primordial dwarfism and immunodeficiency. These studies suggest the importance of dissecting the mechanisms involved in the functioning of replicative DNA polymerases in understanding and treating a range of human diseases. Previous studies in Drosophila have established this organism as a useful model to understand a variety of human diseases. Here, we review the studies on Drosophila that explored the link between DNA polymerases and human disease. First, we summarize the recent studies linking replicative DNA polymerases to various human diseases and disorders. We then review studies on replicative DNA polymerases in Drosophila. Finally, we suggest the possible use of Drosophila models to study human diseases and disorders associated with replicative DNA polymerases.

Quantitative proteomics analysis in small cell carcinoma of cervix reveals novel therapeutic targets

BackgroundAs a rare pathologic subtype, small cell carcinoma of the cervix (SCCC) is characterized by extensive aggressiveness and resistance to current therapies. To date, our knowledge of SCCC origin and progression is limited and sometimes even controversial. Herein, we explored the whole-protein expression profiles in a panel of SCCC cases, aiming to provide more evidence for the precise diagnosis and targeting therapy.MethodsEighteen SCCC samples and six matched normal cervix tissues were collected from January 2013 to December 2017. Data independent acquisition mass spectrometry (DIA) was performed to discriminate the different proteins (DEPs) associated with SCCC. The expression of CDN2A and SYP in corresponding SCCC tissues was verified using immunohistochemistry. GO and KEGG enrichment analyses were used to identify the key DEPs related to SCCC development and tumor recurrence.ResultsAs a result, 1311 DEPs were identified in SCCC tissues (780 up-regulated and 531 down-regulated). In up-regulated DEPs, both GO analysis and KEGG analysis showed the most enriched were related to DNA replication (including nuclear DNA replication, DNA-dependent DNA replication, and cell cycle DNA replication), indicating the prosperous proliferation in SCCC. As for the down-regulated DEPs, GO analysis showed that the most enriched functions were associated with extracellular matrix collagen-containing extracellular matrix. KEGG analysis revealed that the DEPs were enriched in Complement and coagulation cascades, proteoglycans in cancer, and focal adhesion-related pathways. Down-regulation of these proteins could enhance the mobility of cancer cells and establish a favorable microenvironment for tumor metastasis, which might be accounted for the frequent local and distant metastasis in SCCC. Surprisingly, the blood vessels and circulatory system exhibit a down-regulation in SCCC, which might be partly responsible for its resistance to anti-angiogenic regimens. In the stratification analysis of early-stage tumors, a group of enzymes involved in the cancer metabolism was discriminated in these recurrence cases.ConclusionsUsing quantitative proteomics analysis, we first reported the whole-protein expression profiles in SCCC. Significant alterations were found in proteins associated with the enhancement of DNA replication and cellular motility. Besides the association with mitosis, a unique metabolic feature was detected in cases with tumor recurrence. These findings provided novel targets for disease surveillance and treatments, which warranted further validation in the future.

Abstract 6556: A gene signature of DNA damage response pathway is predictive of patient clinical outcomes and is associated with tumor immune microenvironment of lung adenocarcinoma

Abstract DNA damage response (DDR) is crucial to prevent the accumulation of DNA lesions that can lead to genomic instability and promote tumorigenesis. Dysregulation of DDR pathways also provides therapeutic opportunities for new cancer therapy. Given that lung cancer displays a high level of genetic mutation and genomic instability, a comprehensive understanding of the dysregulation of DDR genes in lung cancer, especially non-small cell lung cancer (NSCLC) which accounts for 85% of all lung cancer cases, will be critical for identifying new genes contributing to tumorigenesis and novel targets for lung cancer therapy. The goal of this study is to identify novel targets for lung cancer treatment through a comprehensive analysis of DDR genes and its association with patient survival, genomic mutation/instability, tumor immune microenvironment (TIME), and tumor proliferation ability. To this end, we have comprehensively characterized the expression of 276 DDR genes using the Cancer Genome Atlas (TCGA) and other publicly available data of lung adenocarcinoma (LUAD), the most common NSCLC. We found that 36 out of 276 DDR genes are dysregulated with majority being upregulated in tumors and are associated with patient overall survival (OS) and progression free survival (PFS) based on the TCGA LUAD cohort. A gene expression signature, i.e., DDR gene expression dysregulation score (DDRDS), was constructed based on the geometric mean of the differentially expressed genes that were also associated with patient OS. Patients with a higher DDRDS showed significantly worse OS and PFS, more advanced tumor stage, a higher tumor mutational burden (TMB), neoantigen load, and homologous recombination deficiency (HRD) score. In addition, tumors with higher DDRDS displayed an increase in CD4+ activated memory T-cells, CD8+ T- cells, M0 and M1 macrophage, and NK cells, but a decrease in B cells, CD4+ resting memory T-cells, monocytes, and dendritic cells in TIME. Further, these tumors also showed a higher expression of immune check point molecules PD1 and PD-L1. Lastly, gene set enrichment analysis demonstrated that these tumors had significantly increased nuclear division, DNA replication, chromosome segregation, and cell cycles, reflecting the aggressiveness of the disease. Hence, despite the previous studies demonstrating that mutation and deletion defects in DDR genes are frequent in tumors, we found that the dysregulated DDR genes are mainly upregulated, and DDRDS based on the dysregulated expression of DDR genes is associated with the alteration in TMB/HRD/TIME, tumor aggressiveness, and ultimately patient outcomes. These results suggested that patients with a higher tumor DDRDS are associated with poor prognosis but might benefit from immune checkpoint blockade-associated immunotherapy. Citation Format: Xiaoli Zhang, Junran Zhang. A gene signature of DNA damage response pathway is predictive of patient clinical outcomes and is associated with tumor immune microenvironment of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6556.

The Dual Role of ZEB2 in COAD Metastasis and Immunology

Objective: The zinc finger E-box binding homeobox (ZEB2), which can accelerate the nuclear DNA replication by inducing the activation of upstream transcription promoters, was widely considered as an oncogene. Recent study has found that the overexpression of ZEB2 is associated with a better prognosis in hepatocellular carcinoma. However, its roles in tumor growth, metastasis, and immunology are yet to be elucidated in COAD. Methods: The pan-cancer sequencing data was acquired from The Cancer Genome Atlas (TCGA)-Pan cancer cohort, normal human tissue data was acquired from the Genotype-tissue expression (GTEx) database, and Broad Institute Cancer Cell Line Encyclopedia (CCLE) were downloaded from UCSC Xena. We used the cBioPortal webtool to analyze and visualize the ZEB2 pan-cancer genomic alteration rate. GEO Expression Datasets were used to explore ZEB2 expression levels in COAD patients. UCSC Xena database was used to download prognostic information of COAD patients. The Cox regression and Kaplan–Meier analyses were used to assess the prognostic role of ZEB2 in COAD. Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to determine the biological pathways. Gene Ontology (GO) enrichment analysis was performed to determine the biological processes, molecular functions, and cellular components that were altered in a ZEB2-dependent manner in COAD. The module analysis of PPI interaction network was performed using the MCODE tool of Cytoscape software, and the characteristic molecules were selected by cytohHubba tool. CIBERSORTx database was used to analyze the ZEB2 expression in the presence of 22 types of immune infiltrating cells. Results: This study found that ZEB2 was aberrantly expressed in most cancer types, and it was significantly downregulated in COAD compared with normal tissue. In addition, our findings also show that overexpression of ZEB2 was associated with a better prognosis in COAD. Mechanistic analysis revealed that overexpression of ZEB2 was associated with the neutrophil extracellular trap formation in COAD. And the results show that ZEB2 expression was significantly correlated with several kinds of immune cell infiltration. Conclusion: This study demonstrates that overexpression of ZEB2 was associated with better prognoses in patients with COAD. ZEB2 has close relationship with ACTB, which was highly related to NETs. These findings suggest a dual role of ZEB2 in COAD growth, metastasis, and immunology.

Dual roles for CND1 in maintenance of nuclear and chloroplast genome stability in plants

The coordination of chloroplast and nuclear genome status is critical for plant cell function. Here, we report that Arabidopsis CHLOROPLAST AND NUCLEUS DUAL-LOCALIZED PROTEIN 1 (CND1) maintains genome stability in the chloroplast and the nucleus. CND1 localizes to both compartments, and complete loss of CND1 results in embryo lethality. Partial loss of CND1 disturbs nuclear cell-cycle progression and photosynthetic activity. CND1 binds to nuclear pre-replication complexes and DNA replication origins and regulates nuclear genome stability. In chloroplasts, CND1 interacts with and facilitates binding of the regulator of chloroplast genome stability WHY1 to chloroplast DNA. The defects in nuclear cell-cycle progression and photosynthesis of cnd1 mutants are respectively rescued by compartment-restricted CND1 localization. Light promotes the association of CND1 with HSP90 and its import into chloroplasts. This study provides a paradigm of the convergence of genome status across organelles to coordinately regulate cell cycle to control plant growth and development.

In Situ Analysis of Mitochondrial DNA Synthesis Using Metabolic Labeling Coupled to Fluorescence Microscopy.

Metabolic labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) enables the selective labeling of DNA synthesis in live cells. Newly synthesized EdU-containing DNA can be covalently modified after extraction or in fixed cells using copper-catalyzed azide-alkyne cycloaddition "click chemistry" reactions, enabling bioconjugation to various substrates including fluorophores for imaging studies. While often used to study nuclear DNA replication, EdU labeling can also be leveraged to detect the synthesis of organellar DNA in the cytoplasm of Eukaryotic cells. In this chapter, we outline methods for the application of EdU labeling to the study of mitochondrial genome synthesis in fixed cultured human cells, using fluorescent labeling and superresolution light microscopy.

Prognostic analysis of RAS-related lncRNAs in liver hepatocellular carcinoma.

Liver hepatocellular carcinoma (LIHC), whose incidence is increasing globally, is one of the most prevalent malignant cancers. RAS-related pathways are involved in the cell proliferation, migration, apoptosis, and metabolism in LIHC. Long noncoding RNAs (lncRNAs) also play important roles in the progression and prognosis of LIHC. However, the clinical role, prognostic significance, and immune regulation of RAS-related lncRNAs in LIHC remains unclear. Our study aims to construct and validate a RAS-related lncRNA prognostic risk signature that can estimate the prognosis and response to immunotherapy in LIHC. The clinical information and corresponding messenger RNA (mRNA)/lncRNA expression profiles were obtained from The Cancer Genome Atlas (TCGA) database, and 502 RAS-related lncRNAs were identified by Pearson correlation analysis. A prognostic risk signature with 5 RAS-related lncRNAs was then developed based on the Cox regression and least absolute shrinkage and selection operator (LASSO) algorithm analyses. Subsequently, Kaplan-Meier survival curve, receiver operating characteristic (ROC) curve, and the nomogram were established to evaluate the predictive accuracy of the signature. In addition, the immune microenvironment, tumor mutation burden, and drug sensitivity associated with the signature were also analyzed in LIHC. Compared with the low-risk groups, the high-risk groups had an unfavorable outcome. Multivariate regression analysis revealed that the risk score signature was the independent prognostic factor superior to the other clinical variables. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses demonstrated that the risk score was highly associated with the nuclear division, DNA replication, and immune response. The group with high risk tended to hold a lower immune escape rate and better immunotherapy efficacy, while the group with low risk was more sensitive to some small molecular targeted drugs. We developed a RAS-related lncRNA risk signature that was highly associated with the prognosis and response to immunotherapy and targeted drugs and which provided novel mechanistic insights into the personalized treatment and potential drug selection for patients with LIHC.

Boosting Doxorubicin-Induced Mitochondria Apoptosis for the Monodrug-Mediated Combination of Chemotherapy and Chemodynamic Therapy.

Doxorubicin (Dox)-mediated generation of reactive oxygen radicals (ROS) for mitochondrial apoptosis is identified as a new cytotoxic mechanism in addition to the well-established one via nuclear DNA replication interference. However, this mechanism contributes far less than the latter to Dox therapy. This newly identified pathway to make Dox therapy function like the combination of chemodynamic therapy (CDT) and chemotherapy-mediated by Dox alone would be amplified. One-pot nanoconstruction (HEBD) is fabricated based on the chemical reactions driven assemblies among epigallocatechin gallate (EGCG), buthionine sulfoximine (BSO) and formaldehyde in aqueous mediums followed by Dox adsorption. Acid tumor microenvironments allow the liberation of EGCG, BSO, and Dox due to the breakage of Schiff base bonds. EGCG component in HEBD is responsible for targeting mitochondria and disrupting mitochondrial electron transport chain (mETC) to compel electrons leakage in favor of their capture by Dox to produce more ROS. EGCG-induced mETC disruption results in mitochondrial respiration inhibition with alleviated hypoxia in tumor cells while BSO inhibits glutathione biosynthesis to protect ROS from redox depletion, further boosting Dox-induced CDT. This strategy of amplifying CDT pathway for the Dox-mediated combined therapy could largely improve antitumor effect, extend lifespan of tumor-bearing mice, reduce risks of cardiotoxicity and metastasis.

Molecular Changes in Chronic Myeloid Leukemia During Tyrosine Kinase Inhibitors Treatment. Focus on Immunological Pathways.

IntroductionThe aim of our research was to investigate changes in the molecular background of the immune response in the chronic phase (CP) of chronic myeloid leukaemia (CML) during treatment with tyrosine kinase inhibitors (TKIs).MethodsGlobal gene and miRNA expression profiles were assessed using genome-wide RNA and miRNA microarray technology in bone marrow mononuclear cells. Fifty-one patients were recruited, and bone marrow samples were taken at diagnosis before treatment with TKIs and after 3, 6, and 12 months of treatment with TKIs. The largest number of upregulated genes was observed when the 0-month group (time of diagnosis) was compared to the 3-month group; 1774 genes were significantly upregulated, and 390 genes were significantly downregulated.DiscussionUpregulated biological processes according to gene ontology (GO) classification involved basic cellular processes such as cell division, cell cycle, cell–cell adhesion, protein transport, mitotic nuclear division, apoptosis, and DNA replication. Differentially expressed miRNAs were annotated using GO classification to several immunity-related processes, including the T cell receptor signalling pathway, T cell costimulation, immune response, and inflammatory response. TKI therapy exerts a significant impact on cellular cycle processes and T-cell activation, which was proven at the molecular level.

Efficient replication of human nuclear DNA.

Efficient replication of human nuclear DNA.

Extrinsic proofreading

The high fidelity of replication of the nuclear DNA genome in eukaryotes involves three processes. Correct rather than incorrect dNTPs are almost always incorporated by the three major replicases, DNA polymerases α, δ and ε. When an incorrect base is occasionally inserted, the latter Pols δ and ε also have a 3 ´ to 5 ´ exonuclease activity that can remove the mismatch to allow correct DNA synthesis to proceed. Lastly, rare mismatches that escape proofreading activity and are present in newly replicated DNA can be removed by DNA mismatch repair. In this review, we consider evidence supporting the hypothesis that the second mechanism, proofreading, can operate in two different ways. Primer terminal mismatches made by either Pol δ or Pol ε can be ‘intrinsically’ proofread. This mechanism occurs by direct transfer of a misinserted base made at the polymerase active site to the exonuclease active site that is located a short distance away. Intrinsic proofreading allows mismatch excision without intervening enzyme dissociation. Alternatively, considerable evidence suggests that mismatches made by any of the three replicases can also be proofread by ‘extrinsic’ proofreading by Pol δ. Extrinsic proofreading occurs when a mismatch made by any of the three replicases is initially abandoned, thereby allowing the exonuclease active site of Pol δ to bind directly to and remove the mismatch before replication continues. Here we review the evidence that extrinsic proofreading significantly enhances the fidelity of nuclear DNA replication, and we then briefly consider the implications of this process for evolution and disease.